Genetic locus associated with green snap in maize

ABSTRACT

This invention relates to methods for identifying maize plants that having increased green snap tolerance. The methods use molecular markers to identify and to select plants with increased green snap tolerance or to identify and deselect plants with decreased green snap tolerance. Maize plants generated by the methods of the invention are also a feature of the invention.

This application claims a priority based on provisional application 62/062,475 which was filed in the U.S. Patent and Trademark Office on Oct. 10, 2014, the entire disclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods useful in increasing green snap tolerance in maize plants.

BACKGROUND OF THE INVENTION

Green snap (GS), also called brittle snap, refers to the stalk breakage at or below the ear node when maize is rapidly growing. GS is environmentally dependent. It is hard to predict the severity of the phenomenon. Abiotic factors that are observed to cause GS include strong winds at the most vulnerable growth stage, excessive nitrogen treatment, high temperature and high soil moisture conditions (Elmore, 1999, 2003; Wilhelm, 1999). However, GS is conditioned not only by environmental factors. The genetics of maize also has a significant contribution. It has been observed that certain lines of maize are more tolerant to GS than others.

Green snap could lead to yield loss up to 90% when high wind passes through the corn fields during the rapid growth (elongation) period of vegetative growth. It is not a rare phenomenon. In July 1993 and 1994, severe storms with wind at 100 mph wiped across the western U.S. corn belt, destroyed many corn fields. Almost every other year, green snap causes significant damages in Nebraska, Iowa, Minnesota, Illinois, and periodically in Ohio. However, GS is unpredictable. It depends on both biotic and abiotic factors. The same variety of corn planted a few days apart could have different levels of GS.

Meanwhile, the yield effects of GS may not be as straightforward as suggested by Roger Elmore and Richard B. Ferguson from the University of Nebraska-Lincoln in a linear fashion (based on data from 1993 and 1994): decrease 1% for every 1% increase in stalk breakage. Studies from the University of Minnesota indicate that the grain production of standing plants might compensate for grain loss from broken plants. Depending on where the breakage occurs, stalks broken below the dominant ear have significantly higher yield loss than above the ear with an average of an additional 16 percent less yield.

Green snap has become an increasing issue for seed industries producing high yielding hybrids. The corn hybrids tend to be tall and grow fast, which make them GS-vulnerable. Ironically, the best farming practice, rotating with soybean, tilling, efficient nitrogen use, high moisture and the use of fungicide with growth regulator etc. make the plants more susceptible to the GS. Although genetics have been an effective way to fight against damages from GS, not many studies underlining the resistance has been published. Corn researchers at Pioneer Hi-bred International (PHI) have modified mechanical detasseler to snap corn stalk mimicking the natural GS damage (Barreiro et al, 2011). With its artificial breakage device, PHI has successfully selected high yield hybrids with moderate to high level of GS tolerance. In 1999, Benson presented his QTL analysis in an elite non-stiff stalk breeding population in the 63^(rd) Corn and Sorghum Research Conference. In his study, three QTLs explained 66% of the genetic variation. However, no further validation study has been published so far.

SUMMARY OF THE INVENTION

In one embodiment, methods of identifying a maize plant that displays increased GS tolerance, comprising detecting in germplasm of the maize plant at least one allele of a locus are provided. The GS-resistance locus is located within an interval of the chromosome 3 flanked by PZE-103082924 and PZE-103101055 single nucleotide polymorphism (SNP) markers; and at least one allele is associated with increased GS tolerance. The locus can be selected from any of the following marker loci PZE-103082924, PZE-103091780, PZE-103092640, PZE-103092849, PZE-103095737, PZE-103095917, PZE-103096687, PZE-103097315, PZE-103097436, PZE-103099284, PZE-103099912, PZE-103099943, PZE-103100139, PZE-103100805 and PZE-103101055, as well as any other marker that is linked to these markers. The marker locus can be found on chromosome 3, within the interval comprising and flanked by PZE-103091780 and PZE-103101055, and comprises at least one allele that is associated with increased GS tolerance. In some embodiments, the maize plant belongs to the Stiff Stalk heterotic group. Maize plants identified by this method are also of interest.

In another embodiment, a method for identifying maize plants with increased GS tolerance by detecting a haplotype in the germplasm of the maize plant is provided. The haplotype comprises alleles at one or more marker loci, wherein the one or more marker loci are found on chromosome 3 within the interval comprising and, flanked by, PZE-103091780 and PZE-103101055. The haplotype comprises alleles at one or more marker loci, wherein the one or more marker loci are found on chromosome 3 and are selected from the group consisting of PZE-103091780, PZE-103092640, PZE-103092849, PZE-103095737, PZE-103095917, PZE-103096687, PZE-103097315, PZE-103097436, PZE-103099284, PZE-103099912, PZE-103099943, PZE-103100139, PZE-103100805, and PZE-103101055. The haplotype is associated with increased GS tolerance.

In a further embodiment, a method of selecting plants with increased GS tolerance is provided. In one aspect, a first maize plant is obtained that has at least one allele of a marker locus wherein the allele is associated with increased GS tolerance. The marker locus can be found on chromosome 3, within the interval comprising and flanked by PZE-103082924 and PZE-103101055. The first maize plant can be crossed to a second maize plant, and the progeny resulting from the cross can be evaluated for the allele of the first maize plant. Progeny plants that possess the allele from the first maize plant can be selected as having increased GS tolerance. Maize plants selected by this method are also of interest.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application. The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2): 345-373 (1984), which are herein incorporated by reference in their entirety. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 contains the PZE-103082924 SNP and flanking sequence.

SEQ ID NO: 2 contains the PZE-103091780 SNP and flanking sequence

SEQ ID NO: 3 contains the PZE-103092640 SNP and flanking sequence.

SEQ ID NO: 4 contains the PZE-103092849 SNP and flanking sequence.

SEQ ID NO: 5 contains the PZE-103095737 SNP and flanking sequence.

SEQ ID NO: 6 contains the PZE-103095917 SNP and flanking sequence.

SEQ ID NO: 7 contains the PZE-103096687 SNP and flanking sequence.

SEQ ID NO: 8 contains the PZE-103097315 SNP and flanking sequence.

SEQ ID NO: 9 contains the PZE-103097436 SNP and flanking sequence.

SEQ ID NO: 10 contains the PZE-103099284 SNP and flanking sequence.

SEQ ID NO: 11 contains the PZE-103099912 SNP and flanking sequence.

SEQ ID NO: 12 contains the PZE-103099943 SNP and flanking sequence.

SEQ ID NO: 13 contains the PZE-103100139 SNP and flanking sequence.

SEQ ID NO: 14 contains the PZE-103100805 SNP and flanking sequence.

SEQ ID NO: 15 contains the PZE-103101055 SNP and flanking sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for identifying and selecting maize plants with increased GS tolerance. The following definitions are provided as an aid to understand the invention.

The term “additive effect” is calculated by the following equation:

Additive effect=(“Elite line effect”−“Donor line effect”)/2.

The higher value of GS phenotype, the more GS susceptible maize plant is.

The lower value of GS phenotype, the more GS resistant maize plant is. The sign of additive effect (negative versus positive) indicates which parent (elite versus donor) contributes to the severity of GS. For example, if additive effect has a positive sign, then QTL controlling GS severity comes from an elite parent, and the allele from donor parent will contribute to the decrease of GS phenotype, i.e. resistance. If additive effect is negative, then QTL controlling GS severity comes from donor parent, and the allele from elite parent will contribute to the decrease of phenotype. In molecular breeding QTL coming from elite (susceptible) parent has little practical value. In this particular case with GS, QTL with positive additive effects have practical value in molecular breeding of GS resistant maize lines.

The term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus.

An “amplicon” is amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).

The term “amplifying” in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid for a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.

The term “assemble” applies to bacterial artificial clones (BACs) and their propensities for coming together to form contiguous stretches of DNA. A BAC “assembles” to a contig based on sequence alignment, if the BAC is sequenced, or via the alignment of its BAC fingerprint to the fingerprints of other BACs. The assemblies can be found using the Maize Genome Browser, which is publicly available on the internet.

An allele is “associated with” a trait when it is linked to it and when the presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.

The “B73 reference genome, version 2” is the physical and genetic framework of the maize B73 genome. It is the result of a sequencing effort utilizing a minimal tiling path of approximately 19,000 mapped BAC clones, and focusing on producing high-quality sequence coverage of all identifiable gene-containing regions of the maize genome. These regions were ordered, oriented, and along with all of the intergenic sequences, anchored to the extant physical and genetic maps of the maize genome. It can be accessed using a genome browser, the Maize Genome Browser, that is publicly available on the internet that facilitates user interaction with sequence and map data.

A “BAC”, or bacterial artificial chromosome, is a cloning vector derived from the naturally occurring F factor of Escherichia coli. BACs can accept large inserts of DNA sequence. In maize, a number of BACs, or bacterial artificial chromosomes, each containing a large insert of maize genomic DNA, have been assembled into contigs (overlapping contiguous genetic fragments, or “contiguous DNA”).

“Backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al., (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross gives rise to the F1 generation: the term “BC1” then refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.

A centimorgan (“cM”) is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.

“Chromosomal interval” designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.

The term “chromosomal interval” designates any and all intervals defined by any of the markers set forth in this invention. A chromosomal interval that correlates with increased GS tolerance is provided. This interval, located on chromosome 3, comprises and is flanked by PZE-103082924 and PZE-103101055. A subinterval of chromosomal interval PZE-103082924 and PZE-103101055 is PZE-103091780 and PZE-103101055.

The term “complement” refers to a nucleotide sequence that is complementary to a given nucleotide sequence, i.e., the sequences are related by the base-pairing rules.

The term “contiguous DNA” refers to overlapping contiguous genetic fragments.

The term “crossed” or “cross” means the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants).

The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant).

The term “crossing” refers to the act of fusing gametes via pollination to produce progeny.

A “favorable allele” is the allele at a particular locus that confers, or contributes to, a desirable phenotype, e.g., increased GS tolerance, or alternatively, is an allele that allows the identification of plants with decreased GS tolerance that can be removed from a breeding program or planting (“counterselection”).

A favorable allele of a marker is a marker allele that segregates with the favorable phenotype, or alternatively, segregates with the unfavorable plant phenotype, therefore providing the benefit of identifying plants.

“Fragment” is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using methods disclosed herein.

A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or chromosomes) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by the recombination frequencies between them, and recombinations between loci can be detected using a variety of molecular genetic markers (also called molecular markers). A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci can differ from one genetic map to another. However, information such as marker position and order can be correlated between maps by determining the physical location of the markers on the chromosome of interest, using the B73 reference genome, version 2, which is publicly available on the internet. One of ordinary skill in the art can use the publicly available genome browser to determine the physical location of markers on a chromosome.

The term “Genetic Marker” shall refer to any type of nucleic acid based marker, including but not limited to, Restriction Fragment Length Polymorphism (RFLP) (Botstein et al, 1998), Simple Sequence Repeat (SSR) (Jacob et al., 1991), Random Amplified Polymorphic DNA (RAPD) (Welsh et al., 1990), Cleaved Amplified Polymorphic Sequences (CAPS) (Rafalski and Tingey, 1993, Trends in Genetics 9:275-280), Amplified Fragment Length Polymorphism (AFLP) (Vos et al, 1995, Nucleic Acids Res. 23:4407-4414), Single Nucleotide Polymorphism (SNP) (Brookes, 1999, Gene 234:177-186), Sequence Characterized Amplified Region (SCAR) (Pecan and Michelmore, 1993, Theor. Appl. Genet, 85:985-993), Sequence Tagged Site (STS) (Onozaki et al. 2004, Euphytica 138:255-262), Single Stranded Conformation Polymorphism (SSCP) (Orita et al., 1989, Proc Natl Aced Sci USA 86:2766-2770). Inter-Simple Sequence Repeat (ISR) (Blair et al. 1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon Amplified Polymorphism (IRAP), Retrotransposon-Microsatellite Amplified Polymorphism (REMAP) (Kalendar et al., 1999, Theor. Appl. Genet 98:704-711), an RNA cleavage product (such as a Lynx tag), and the like. “Genetic recombination frequency” is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and/or traits following meiosis.

“Genome” refers to the total DNA, or the entire set of genes, carried by a chromosome or chromosome set.

The term “genotype” is the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple led, or, more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome.

“Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leafs, stems, pollen, or cells that can be cultured into a whole plant.

“Green Snap” (GS) is a term which describes a corn plant stalk which has been broken by high winds. GS typically occurs at or below the ear, but can occur at any position along the stalk. GS can occur at various developmental stages, but most often occurs between the vegetative stages of V5 and V8 and between the vegetative V12 stage and the reproductive R1 stage.

A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term “haplotype” can refer to sequence, polymorphisms at a particular locus, such as a single marker locus, or sequence polymorphisms at multiple loci along a chromosomal segment in a given genome. The former can also be referred to as “marker haplotypes” or “marker alleles”, while the latter can be referred to as “long-range haplotypes”.

The “heritability (h²)” of a trait within a population is the proportion of observable differences in a trait between individuals within a population that is due to genetic differences. The h² value of the QTL is a percentage of variation that is explained by genetics, instead of environment.

A “heterotic group” comprises a set of genotypes that result in heterosis when crossed with genotypes from a different heterotic group (Hallauer at al. (1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley (ed) Corn and corn improvement). Inbred lines are classified into heterotic groups, and are further subdivided into families within a heterotic group, based on several criteria such as pedigree, molecular marker-based associations, and performance in hybrid combinations (Smith at al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely used heterotic groups in the United States are referred to as “Iowa Stiff Stalk Synthetic” (BSSS) and “Lancaster” or “Lancaster Sure Crop” (sometimes referred to as NSS, or Iron-Stiff Stalk).

The term “heterozygous” means a genetic condition wherein different alleles reside at corresponding loci on homologous chromosomes.

The term “homozygous” means a genetic condition wherein identical alleles reside at corresponding loci on homologous chromosomes.

“Hybridization” or “nucleic acid hybridization” refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.

The term “hybridize” means the formation of base pairs between complementary regions of nucleic acid strands.

The term “indel” refers to an insertion or deletion, wherein one line may be referred to as having an insertion relative to a second line, or the second line may be referred to as having a deletion relative to the first line.

The term “introgression” or “introgressing” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a selected allele of a marker, a QTL, a transgene, or the like. In any case, offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background. For example, the chromosome 3 locus described herein may be introgressed into a recurrent parent that has problematic GS. The recurrent parent line with the introgressed gene or locus then has increased GS tolerance.

As used herein, the term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus or some other locus (for example, a GS locus). The linkage relationship between a molecular marker and a phenotype is given as a “probability” or “adjusted probability”. Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units for cM). In some aspects, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes. Thus, “closely linked loci” such as a marker locus and a second locus display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10 (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” each other. Since one cM is the distance between two markers that show a 1% recombination frequency, any marker is closely linked (genetically and physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distant. Two closely linked markers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each other.

The term “linkage disequilibrium” refers to a non-random segregation of genetic loci or traits for both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency (in the case of co-segregating traits, the loci that underlie the traits are in sufficient proximity to each other). Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51% to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same chromosome.) As used herein, linkage can be between two markers, or alternatively between a marker and a phenotype. A marker locus can be “associated with” (linked to) a trait, e.g., increased GS tolerance. The degree of linkage of a molecular marker to a phenotypic trait is measured, e.g. as a statistical probability of co-segregation of that molecular marker with the phenotype.

Linkage disequilibrium is most commonly assessed using the measure r², which is calculated using the formula described by Hill, W. G. and Robertson, A, Theor Appl. Genet 38:226-231 (1988). When r²=1, complete LD exists between the two marker loci, meaning that the markers have not been separated by recombination and have the same allele frequency. Values for r² above ⅓ indicate sufficiently strong LD to be useful for mapping (Ardlie at al., Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are in linkage disequilibrium when r² values between pairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

As used herein, “linkage equilibrium” describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).

The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science 255:803-804 (1992)) is used in interval mapping to describe the degree of linkage between two marker loci. A LOD score of three between two markers indicates that linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage. LOD scores greater than or equal to two may be used to detect linkage.

A “locus” is a position on a chromosome where a gene or marker is located.

“Maize” refers to a plant of the Zea mays L. ssp. mays and is also known as “corn”.

The term “maize plant” includes: whole maize plants, maize plant cells, maize plant protoplast, maize plant cell or maize tissue cultures from which maize plants can be regenerated, maize plant calli, and maize plant cells that are intact in maize plants or parts of maize plants, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips, and the like.

A “marker” is a nucleotide sequence or encoded product thereof (e.g., a protein) used as a point of reference. For markers to be useful at detecting recombinations, they need to detect differences, or polymorphisms, within the population being monitored. For molecular markers, this means differences at the DNA level due to polynucleotide sequence differences (e.g. SSRs, RFLPs, AFLPs, SNPs). The genomic variability can be of any origin, for example, insertions, deletions, duplications, repetitive elements, point mutations, recombination events, or the presence and sequence of transposable elements. Molecular markers can be derived from genomic or expressed nucleic acids (e.g., ESTs) and can also refer to nucleic acids used as probes or primer pairs capable of amplifying sequence fragments via the use of PCR-based methods. A large number of maize molecular markers are known in the art, and are published or available from various sources, such as the Maize GDB Internet resource and the Arizona Genomics Institute Internet resource run by the University of Arizona.

Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art. These include, e.g., DNA sequencing, PCR-based sequence specific amplification methods, detection of RFLPs, detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of SSRs, detection of SNPs, or detection of FLPs. Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and RAPDs.

A “marker allele”, alternatively an “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population that is polymorphic for the marker locus.

“Marker assisted selection” (or MAS) is a process by which phenotypes are selected based on marker genotypes.

“Marker assisted counter-selection” is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.

A “marker locus” is a specific chromosome location in the genome of a species when a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., a linked locus that encodes or contributes to expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a locus, such as a QTL or single gene, that are genetically or physically linked to the marker locus.

A “marker probe” is a nucleic add sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic add hybridization. Marker probes comprising 30 or more contiguous nucleotides of the marker locus (“all or a portion” of the marker locus sequence) may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e. genotype) the particular allele that is present at a marker locus.

The term “molecular marker” may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A “molecular marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are “complementary” when they specifically hybridize in solution, e.g., according to Watson-Crick base pairing rules. Some of the markers described herein are also referred to as hybridization markers when located on an indel region, such as the non-collinear region described herein. This is because the insertion region is, by definition, a polymorphism vis a via a plant without the insertion. Thus, the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g., SNP technology is used in the examples provided herein.

“Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and “nucleic acid fragment” are used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A “nucleotide” is a monomeric unit from which DNA or RNA polymers are constructed, and consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate. “G” for guanylate or deoxyguanylate. “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

The terms “phenotype”, or “phenotypic trait” or “trait” refers to one or more traits of an organism. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait”. In other cases, a phenotype is the result of several genes.

A “physical map” of the genome is a map showing the linear order of identifiable landmarks (including genes, markers, etc.) on chromosome DNA. However, in contrast to genetic maps, the distances between landmarks are absolute (for example, measured in base pairs or isolated and overlapping contiguous genetic fragments) and not based on genetic recombination.

A “plant” can be a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same. A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.

A “polymorphism” is a variation in the DNA that is too common to be due merely to new mutation. A polymorphism must have a frequency of at least 1% in a population. A polymorphism can be a single nucleotide polymorphism, or SNP, or an insertion/deletion polymorphism, also referred to herein as an “indel”.

The “probability value” or “p-value” is the statistical likelihood that the particular combination of a phenotype and the presence or absence of a particular marker allele is random. Thus, the lower the probability score, the greater the likelihood that a phenotype and a particular marker will co-segregate. In some aspects, the probability score is considered “significant” or “nonsignificant”. In some embodiments, a probability score of 0.05 (p=0.05, or a 5% probability) of random assortment is considered a significant indication of co-segregation. However, an acceptable probability can be any probability of less than 50% (p=0.5). For example, a significant probability can be less than 0.25, less than 0.20, less than 0.15, less than 0.1, less than 0.05, less than 0.01, or less than 0.001.

The term “progeny” refers to the offspring generated from a cross.

A “progeny plant” is generated from a cross between two plants.

Each “PZE” SNP assay represents a DAS proprietary SNP marker that was developed using the Infinium iSelect® chemistry (Illumina, San Diego, Calif.).

A “reference sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence is obtained by genotyping a number of lines at the locus, aligning the nucleotide sequences in a sequence alignment program (e.g. Sequencher), and then obtaining the consensus sequence of the alignment.

A “single nucleotide polymorphism (SNP)” is an allelic single nucleotide—A, T, C or G—variation within a DNA sequence representing one locus of at least two individuals of the same species. For example, two sequenced DNA fragments representing the same locus from at least two individuals of the same species, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide.

The “Stiff Stalk” heterotic group represents a major heterotic group in the northern U.S. and Canadian corn growing regions. It can also be referred to as the Iowa Stiff Stalk Synthetic for BSSS heterotic group.

A “topcross test” is a progeny test derived by crossing each parent with the same tester, usually a homozygous line. The parent being tested can be an open-pollinated variety, a cross, or an inbred line.

The phrase “under stringent conditions” refers to conditions under which a probe or polynucleotide will hybridize to a specific nucleic acid sequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances.

Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50 of the probes are occupied at equilibrium), Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium on concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as form amide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions are often: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SOS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C., depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references.

Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal V method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10), Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic adds these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

Before describing the present invention in detail, it should be understood that this invention is not limited to particular embodiments. It also should be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting. As used herein and in the appended claims, terms in the singular and the singular forms “a”, “an” and “the”, for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “plant”, “the plant” or “a plant” also includes a plurality of plants. Depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant. The use of the term “a nucleic acid” optionally includes many copies of that nucleic acid molecule.

Genetic Mapping

It has been recognized for quite some time that specific genetic loci correlating with particular phenotypes, such as reduced GS, can be mapped in an organism's genome. The plant breeder can advantageously use molecular markers to identify desired individuals by detecting marker alleles that show a statistically significant probability of co-segregation with a desired phenotype, manifested as linkage disequilibrium. By identifying a molecular marker or clusters of molecular markers that co-segregate with a trait of interest, the breeder is able to rapidly select a desired phenotype by selecting for the proper molecular marker allele (a process called marker-assisted selection, or MAS).

A variety of methods well known in the art are available for detecting molecular markers or clusters of molecular markers that co-segregate with a trait of interest, such as increased GS tolerance. The basic idea underlying these methods is the detection of markers, for which alternative genotypes (or alleles) have significantly different average phenotypes. Thus, one makes a comparison among marker loci of the magnitude of difference among alternative genotypes (or alleles) or the level of significance of that difference. Trait genes are inferred to be located nearest the marker(s) that have the greatest associated genotypic difference.

Two such methods used to detect trait loci of interest are: 1) Population-based association analysis and 2) Traditional linkage analysis. In a population-based association analysis, lines are obtained from pre-existing populations with multiple founders, e.g. elite breeding lines. Population-based association analyses rely on the decay of linkage disequilibrium (LD) and the idea that in an unstructured population, only correlations between genes controlling a trait of interest and markers closely linked to those genes will remain after so many generations of random mating. In reality, most pre-existing populations have population substructure. Thus, the use of a structured association approach helps to control population structure by allocating individuals to populations using data obtained from markers randomly distributed across the genome, thereby minimizing disequilibrium due to population structure within the individual populations (also called subpopulations). The phenotypic values are compared to the genotypes (alleles) at each, marker locus for each line in the subpopulation. A significant marker-trait association indicates the dose proximity between the marker locus and one or more genetic loci that are involved in the expression of that trait.

The same principles underlie traditional linkage analysis; however, LD is generated by creating a population from a small number of founders. The founders are selected to maximize the level of polymorphism within the constructed population, and polymorphic sites are assessed for their level of cosegregation with a given phenotype. A number of statistical methods have been used to identify significant marker-trait associations. One such method is an interval mapping approach (Lander and Botstein, Genetics 121:185-199 (1989), in which each of many positions along a genetic map (say at 1 cM intervals) is tested for the likelihood that a gene controlling a trait of interest is located at that position. The genotype/phenotype data are used to calculate for each test position a LOD score (log of likelihood ratio). When the LOD score exceeds a threshold value, there is significant evidence for the location of a gene controlling the trait of interest at that position on the genetic map (which will fall between two particular marker loci).

Markers Associated with Green Snap Tolerance

Markers associated with GS tolerance are identified herein. The methods involve detecting the presence of at least one marker allele associated with the enhanced resistance in the germplasm of a maize plant. The marker locus can be selected from any of the marker loci provided in Table 2, including PZE-103091780, PZE-103092640, PZE-103092849, PZE-103095737, PZE-103095917, PZE-103096687, PZE-103097315, PZE-103097436, PZE-103099284, PZE-103099912, PZE-103099943, PZE-103100139, PZE-103100805 and PZE-103101055, and any other marker linked to these markers (linked markers can be determined from the MaizeGDB resource). The marker locus can be selected from any of the marker loci provided in Table 2, including PZE-103082924 and any other marker linked to this marker (linked markers can be determined from the Maize GDB resource).

The genetic elements or genes located on a contiguous linear span of genomic DNA on a single chromosome are physically linked. PZE-103082924 and PZE-103101055, both highly associated with GS tolerance, delineate a GS tolerance QTL. Any polynucleotide that assembles to the contiguous DNA between and including SEQ ID NO:1 (the reference sequence for PZE-103082924), or a nucleotide sequence that is 95% identical to SEQ ID NO:1 based on the Clustal V method of alignment, and SEQ ID NO:15 (the reference sequence for PZE-103101055), or a nucleotide sequence that is 95% identical to SEQ ID NO:15 based on the Clustal V method of alignment, can house marker loci that are associated with GS tolerance.

The genetic elements or genes located on a contiguous linear span of genomic DNA on a single chromosome are physically linked for the subinterval of PZE-103091780 and PZE-103101055. PZE-103091780 and PZE-103101055, both highly associated with GS tolerance, delineate a GS tolerance QTL. Any polynucleotide that assembles to the contiguous DNA between and including SEQ ID NO:3 (the reference sequence for PZE-103091780), or a nucleotide sequence that is 95% identical to SEQ ID NO:3 based on the Clustal V method of alignment, and SEQ ID NO:15 (the reference sequence for PZE-103101055), or a nucleotide sequence that 95% identical to SEQ ID NO:15 based on the Clustal V method of alignment, can house marker loci that are associated with increased GS tolerance.

A common measure of linkage is the frequency with which traits cosegregate. This can be expressed as a percentage of cosegregation (recombination frequency) or in centiMorgans (cM). The cM is a unit of measure of genetic recombination frequency. One cM is equal to a 1% chance that a trait at one genetic locus will be separated from a trait at another locus due to crossing over in a single generation (meaning the traits segregate together 99% of the time). Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.

Marker loci are themselves traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus, due to crossing over in a single generation.

Other markers linked to the markers listed in Table 2 can be used to predict GS tolerance in a maize plant. This includes any marker within 50 cM of PZE-103082924, PZE-103091780, PZE-103092640, PZE-103092849, PZE-103095737, PZE-103095917, PZE-103096687, PZE-103097315, PZE-103097436, PZE-103099284, PZE-103099912, PZE-103099943, PZE-103100139, PZE-103100805, and PZE-103101055, the markers associated with the GS tolerance. The closer a marker is to a gene controlling a trait of interest, the more effective and advantageous that marker is as an indicator for the desired trait. Closely linked loci display an inter-locus cross-over frequency of about 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci (e.g., a marker locus and a target locus) display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8% 7%, 6%, 5%, 4%, 3%, 2% 1%, 0.75%, 0.5%, 0.25.degree., or less) are said to be “proximal to” each other.

Although particular marker alleles can show co-segregation with increased GS tolerance, it is important to note that the marker locus is not necessarily responsible for the expression of the GS tolerance phenotype. For example, it is not a requirement that the marker polynucleotide sequence be part of a gene that imparts increased GS tolerance (for example, is part of the gene open reading frame). The association between a specific marker allele and the increased GS tolerance phenotype is due to the original “coupling” linkage phase between the marker allele and the allele in the ancestral maize line from which the allele originated. Eventually, with repeated recombination, crossing over events between the marker and genetic locus can change this orientation. For this reason, the favorable marker allele may change depending on the linkage phase that exists within the resistant parent used to create segregating populations. This does not change the fact that the marker can be used to monitor segregation of the phenotype. It only changes which marker allele is considered favorable in a given segregating population.

The term “chromosomal interval” designates any and all intervals defined by any of the markers set forth in this invention. A chromosomal interval that correlates with GS tolerance is provided. This interval, located on chromosome 3, comprises and is flanked by PZE-103082924 and PZE-103101055. A subinterval of chromosomal interval PZE-103082924 and PZE-103101055 is PZE-103091780 and PZE-103101055.

A variety of methods well known in the art are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers that will be linked to the gene controlling the trait of interest. In other words, the chromosomal interval is drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for GS tolerance. The interval described above encompasses a cluster of markers that co-segregate with GS tolerance. The clustering of markers occurs in relatively small domains on the chromosomes, indicating the presence of a gene controlling the trait of interest in those chromosome regions. The interval was drawn to encompass the markers that co-segregate with GS tolerance. The interval encompasses markers that map within the interval as well as the markers that define the termini. For example, PZE-103082924 and PZE-103101055, separated by 24,122,049 bp based on the B73 reference genome, version 2, define a chromosomal interval encompassing a cluster of markers that co-segregate with GS tolerance. A second example includes the subinterval, PZE-103091780 and PZE-103101055, separated by 10,015,643 bp based on the B73 reference genome, version 2, that defines a chromosomal interval encompassing a cluster of markers that co-segregate with GS tolerance. An interval described by the terminal markers that define the endpoints of the interval will include the terminal markers and any marker localizing within that chromosomal domain, whether those markers are currently known or unknown.

Chromosomal intervals can also be defined by markers that are linked to (show linkage disequilibrium with) a marker of interest, and is a common measure of linkage disequilibrium (LD) in the context of association studies. If the r² value of LD between any chromosome 3 marker locus lying within the interval of PZE-103082924 and PZE-10310105, the subinterval of PZE-103091780 and PZE-103101055, or any other subinterval of PZE-103082924 and PZE-103101055, and an identified marker within that interval that has an allele associated with increased GS tolerance is greater than ⅓ (Ardlie et al. Nature Reviews Genetics 3:299-309 (2002)), the loci are linked.

A marker of the invention can also be a combination of alleles at marker loci, otherwise known as a haplotype. The skilled artisan would expect that there might be additional polymorphic sites at marker loci in and around the chromosome 3 markers identified herein, wherein one, or more polymorphic sites is in linkage disequilibrium (LD) with an allele associated with increased GS tolerance. Two particular alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)).

Marker Assisted Selection

Molecular markers can be used in a variety of, plant breeding applications (e.g. see Staub et al. (1996) Hortscience 729-741; Tanksley (1983) Plant Molecular Biology Reporter 1: 3-8). One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS). A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true with traits that are difficult to phenotype due to their dependence on environmental conditions. This category includes traits related to the resistance to biotic and abiotic stresses. This category also includes traits that are very expensive to phenotype because of laborious artificial inoculation or maintenance of managed stress environments. Another category of traits includes those, which are associated with destruction of the plant per se. Destructive phenotyping has been a bottleneck to implement MAS for seed quality traits. As DNA marker assays are environmentally independent, robust, reliable, less laborious, less costly and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. The ideal situation is to have a marker in the gene itself, so that recombination cannot occur between the marker and the gene. Such a marker is called a ‘perfect marker’.

When a gene is introgressed by MAS, it is not only the gene that is introduced but also the flanking regions (Gepts. (2002). Crop Sci; 42: 1780-1790). This is referred to as “linkage drag.” In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. This “linkage drag” may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite maize line. This is also sometimes referred to as “yield drag.” The size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al, (1998) Genetics 120:579-585). In classical breeding it is usually only by chance that recombinations are selected that contribute to a reduction in the size of the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near the gene of interest. In 150 backcross plants, there is a 95% chance that at least one plant will have experienced a crossover within 1 cM of the gene, based on a single meiosis map distance. Markers will avow unequivocal identification of those individuals. With one additional backcross of 300 plants, there would be a 95% chance of a crossover within 1 cM single meiosis map distance of the other side of the gene, generating a segment around the target gene of less than 2 cM based on a single meiosis map distance. This can be accomplished in two generations with, markers, while it would have required on average 100 generations without markers (See Tanksley et al., supra). When the exact location of a gene is known, flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.

The availability of the B73 reference genome, version 2 and the integrated linkage maps of the maize genome containing increasing densities of public maize markers, has facilitated maize genetic mapping and MAS. See, e.g. the IBM2 Neighbors maps, which are available online on the Maize GDB website.

The key components to the implementation of MAS are (i) defining the population within which the marker-trait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and/or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.

SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6). Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396), SSRs are highly suited to mapping and MAS as they are multi-allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al. (1996) Generating and using DNA markers in plants. In Non-mammalian genomic analysis: a practical guide. Academic Press, pp 75-135).

Various types of SSR markers can be generated, and SSR profiles from resistant lines can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment. An SSR service for maize is available to the public on a contractual basis by DNA Landmarks in Saint-Jean-sur-Richelieu, Quebec, Canada.

Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus having the potential to provide the highest genetic map resolution (Bhattramakki et al. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called ‘ultra-high-throughput’ fashion, as they do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp, 475-492: Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100: Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R, J Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, VVallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode™. (Qiagen), Invader® (Third Wave Technologies), SnapShot® (Applied Biosystems), Taqman® (Applied Biosystems) and Beadarrays™ (Illumina).

A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than, single SNPs and can be more descriptive of any particular genotype. For example, single SNP may be allele ‘T’ for a specific line or variety with increased GS tolerance, but the allele ‘T’ might also occur in the maize breeding population being utilized for recurrent parents. In this case, a haplotype, e.g. a combination of alleles at linked SNP markers, may be more informative. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. See, for example, WO2003054229. Using automated high throughput marker detection platforms known to those of ordinary skill in the art makes this process highly efficient and effective.

The sequences listed in Table 2 can be readily used to obtain additional polymorphic SNPs (and other markers) within the QTL interval listed in this disclosure. Markers within the described map region can be hybridized to BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.

In addition to SSR's, FLPs, and SNPs, as described above, other types of molecular markers are also widely used, including but not limited to, SSR markers derived from EST sequences, RAPD, and other nucleic acid based markers.

Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and/or genetic maps are aligned to locate equivalent markers not described within this disclosure but that are within similar regions. These maps may be within the maize species, or even across other species whose genomes share some level of colinearity at macro- and micro-level with maize, such as rice, and sorghum.

In general, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with GS tolerance. Such markers are presumed to map near quantitative trait loci (QTL) give the plant its GS tolerance phenotype, and are considered indicators for the desired trait, or markers. Markers test maize plants for the presence of a desired allele, and those which contain a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. The means to identify maize plants that have increased GS tolerance by identifying plants that have a specified allele at any one of marker loci described herein, including PZE-103082924, PZE-103091780, PZE-103092640, PZE-103092849, PZE-103095737, PZE-103095917, PZE-103096687, PZE-103097315, PZE-103097436, PZE-103099284, PZE-103099912, PZE-103099943, PZE-103100139, PZE-103100805 and PZE-103101055 are presented herein.

The interval presented herein finds use in MAS to select plants that demonstrate increased GS tolerance. Any marker that maps within the chromosome 3 interval defined by and including PZE-103082924 and PZE-103101055 can be used for this purpose. In addition, haplotypes comprising alleles at one or more marker loci within the chromosome 3 interval defined by and including PZE-103082924 and PZE-103101055 can be used to introduce increased GS tolerance into maize lines or varieties. Any allele or haplotype that is in linkage disequilibrium with an allele associated with increased GS tolerance can be used in MAS to select plants with increased GS tolerance.

EXAMPLES

The following examples are offered to illustrate, but not to limit, the appended claims. It is understood that the examples and embodiments described herein are for illustrative purposes only and that persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the invention or the scope of the appended claims.

Example 1 Genetic Materials

Seventy-five double haploid (DH) lines of a population developed from a cross between ‘STC13’×‘BE4207’ were planted (10-12 kernels/line per row) in two environments, Davenport, Iowa and Mount Vernon, Ind. in 2011. STC13 is an adapted Argentinean inbred line and tolerant to GS. BE4207 is a Dow AgroSciences (DAS) high impact inbred line with an excellent general combining ability. However, BE4207 is highly susceptible to GS. Leaf punches were collected in July 2011, from Sidney and extracted with a standard MagAttract 96 DNA Plant Core Kit (Qiagen, Valencia, Calif.) with a customized BioCel robot system from Agilent Technologies (Santa Clara, Calif.). Prior to PCR, DNA samples were quantified with Quant-iT™ PicoGreen® Quantification Kit (Invitrogen, Carlsbad, Calif.) using manufacturer's instructions. DNA from each sample were sequenced with the HiSeq 2000 (Illumina, San Diego, Calif.) using 10× coverage and standard protocols. SNP genotyping for each DNA sample (200 ng/sample) was performed using DAS custom Infinium iSelect (Illumina, San Diego, Calif.).

SNP genotyping data from the Infinium iSelects were analyzed with GenomeStudio 2011.1 software (Illumina, San Diego, Calif.) and a total of 13,972 polymorphic SNPs were identified. The iSelect SNPs are referred to as the iSelect dataset.

Example 2 Phenotype Data Collection

Phenotypic data were collected from two locations: Davenport and Mount Vernon in August 2011. Each line had 10 to 12 plants in a row. Phenotype was based on naturally occurred snapping. The GS severity rating was taken from the entire row and was represented as a ratio of snapped plants to the total number of plants per row. For example, if three plants were snapped out of 12 in total, then GS severity would be 3/12.

As the phenotypic data collected from Davenport and Mount Vernon were based on the naturally occurred stalk breakage at both locations, there were many lines with no GS, which were either GS tolerant lines or false positive escapes. Since GS is highly environmentally dependant, escape is hard to avoid when the occurrence is from natural wind damage. However, using restricted maximum likelihood (REML) estimation of variance in JMP 9.0 (SAS, Cary, N.C.) revealed a low environment effect with 14.74% (Table 1). The heritability is moderate: 38.66%, and genotype by environment effect is high explaining 46.60% of the variation (Table 1). This is in agreement with the study by Benson (1999), where QTLs accounted for 26% of the total variation.

TABLE 1 REML Variance Component Estimates. Stan- Random Variance Variance dard 95% 95% % of Effect Ratio Component Error Lower Upper Total Line 0.830 0.014 0.004 0.005 0.022 38.662 Site 0.316 0.005 0.008 −0.010 0.021 14.739 Residual 0.017 0.003 0.012 0.025 46.599 Total 0.036 100 −2 Log Likelihood = −80.06640649

Example 3 QTL Mapping

A DAS proprietary Advanced QTL Mapping tool was used to analyze the mapping data. This software consists of two models. The first model employed all of the data, instead of the average, and had an extra factor in the model to estimate the location effect. Location effect estimation accounts for environmental noise in the data and resulted in a better estimate. Traditional Interval Mapping (TIM) and Composite Interval Mapping (CIM) models were employed with a LOD threshold defined based on 1000 permutations of the trait data. The second model implemented extended CIM using GS severity-Location as a covariate.

Based on this analysis, a QTL was identified on chromosome 3 with a LOD score of 4.49. The CIM QTL plot for chromosome 3 shows that the QTL peak occurs at DAS marker PZE-103096687 at 101.76 cM on the DAS proprietary maize map. The QTL interval is flanked by DAS proprietary markers PZE-103082924 and PZE-103101055, spanning a physical distance of 24,122,049 bp based on the B73 reference genome, version 2 (Table 2). Closely linked markers flanking the locus of interest that have alleles in linkage disequilibrium with a favorable allele at that locus may be effectively used to select for progeny plants with increased GS tolerance. Physical locations of the public SNP markers that were close to those described in this invention were determined using the B73 reference genome version 2 on the publicly available Maize GDB website.

The QTL on chromosome 3 has an additive effect of 0.075 (Table 2), indicating that the resistant allele controlling QTL comes from the donor plant. The h² value of the QTL is a percentage of variation that is explained by genetics, instead of environment. The h² value for the QTL on chromosome 3 is 16%, indicating that this QTL is a minor QTL and that there are additional QTL for GS tolerance (Table 2).

TABLE 2 DAS markers significantly associated with green snap tolerance and physical positions based on the B73 reference genome, version 2. Physical Location on Public markers Additive B73 Ref, v2 (bp) DAS Marker from Maize GDB effect h² LOD SEQ ID NO 137,522,228 PZE-103082924 crr5 (aka 0.045 0.09 3.31 1 148,289,177 PZB02044) 151,628,634 PZE-103091780 2 153,261,926 PZE-103092640 0.068 0.12 3.43 3 153,857,820 PZE-103092849 0.071 0.14 3.94 4 155,761,120 PZE-103095737 0.072 0.14 4.01 5 155,803,086 PZE-103095917 0.073 0.147 4.05 6 156,499,588 PZE-103096687 0.075 0.155 4.49^(#) 7 156,963,652 PZE-103097315 0.069 0.131 3.66 8 157,052,742 PZE-103097436 0.070 0.135 3.87 9 158,896,327 PZA00828 159,730,164 PZE-103099284 0.071 0.137 3.85 10 159,873,829 PZE-103099912 0.068 0.127 3.69 11 159,911,178 PZE-103099943 12 160,328,564 PZE-103100139 13 160,755,002 PZE-103100805 14 161,257,755 PHM9914 161,644,277 PZE-103101055 0.064 0.113 3.38 15 ^(#)Significant LOD score at the peak of QTL with significant threshold at LOD = 3.27.

Example 4 Marker Framework and Use for Marker Assisted Selection

A set of common markers can be used to establish a framework for identifying markers in the QTL interval. Table 2 shows markers that are in consistent position relative to one another on the DAS internally derived map and the B73 reference genome, version 2. Physical locations of the DAS proprietary markers were determined using the DAS proprietary GBrowser. The physical locations of public markers that are closely located to DAS proprietary markers on chromosome 3 were determined using the Incongruency tool at Maize GDB.

Closely linked markers flanking the locus of interest that have alleles in linkage disequilibrium with a favorable allele at that locus may be effectively used to select for progeny plants with increased GS tolerance. Thus, the markers described in herein, such as those listed in Table 2 and 3, as well as other markers genetically or physically mapped to the same chromosomal segment, may be used to select for maize plants with increased GS tolerance. Typically, a set of these markers will be used (e.g. 2 or more, 3 or more, 4 or more, 5 or more) in the regions flanking the locus of interest. Optionally, a marker within the actual gene and/or locus may be used.

TABLE 3 Chromosome 3 SNP markers associated with green snap tolerance and their favorable, or donor, allele. SEQ ID Donor DAS Marker NO SNP Allele PZE-103082924 1 A/C C PZE-103091780 2 T/C C PZE-103092640 3 T/C C PZE-103092849 4 A/G G PZE-103095737 5 T/C T PZE-103095917 6 C/G C PZE-103096687 7 T/C T PZE-103097315 8 A/T T PZE-103097436 9 C/G C PZE-103099284 10 T/C T PZE-103099912 11 C/G G PZE-103099943 12 A/G G PZE-103100139 13 A/G A PZE-103100805 14 T/C C PZE-103101055 15 T/C T

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We claim:
 1. A method of identifying a maize plant that displays increased green snap tolerance, the method comprising detecting in germplasm of the maize plant at least one allele of a marker locus wherein: a. the marker locus is located within a chromosomal interval comprising and flanked by PZE-103082924 and PZE-103101055; and b. the at least one allele is associated with increased green snap tolerance.
 2. The method of claim 1, wherein the marker locus is located within a chromosomal interval comprising and flanked by PZE-103091780 and PZE-103101055.
 3. The method of claim 1, wherein the maize plant belongs to the Stiff Stalk heterotic group.
 4. A maize plant identified by the method of claim
 1. 5. A method of identifying a maize plant that displays increased green snap tolerance, the method comprising detecting in germplasm of the maize plant a haplotype comprising alleles at one or more marker loci, wherein: a. the one or more marker loci are located within a chromosomal interval comprising and flanked by PZE-103082924 and PZE-103101055; and, b. the haplotype is associated with increased green snap tolerance.
 6. The method of claim 5, wherein the one or more marker loci are located within a chromosomal interval comprising and flanked by PZE-103082924 and PZE-103101055.
 7. The method of claim 5, wherein the maize plant belongs to the Stiff Stalk heterotic group.
 8. A maize plant identified by the method of claim 5, wherein the maize plant comprises within its germplasm a haplotype associated with increased green snap tolerance wherein the haplotype comprises alleles at one or more marker loci located within a chromosomal interval comprising and flanked by PZE-103082924 and PZE-103101055.
 9. A method of marker assisted selected comprising: a. obtaining a first maize plant having at least one allele of a marker locus, wherein the marker locus is located within a chromosomal interval comprising and flanked by PZE-103082924 and PZE-103101055 and the allele of the marker locus is associated with increased green snap tolerance; b. crossing the first maize plant to a second maize plant; c. evaluating the progeny for the at least one allele; and d. selecting progeny plants that possess the at least one allele.
 10. The method of claim 9, wherein the marker locus is located within a chromosomal interval comprising and flanked by PZE-103091780 and PZE-PZE-103101055.
 11. The method of claim 9, wherein the maize plant belongs to the Stiff Stalk heterotic group.
 12. A maize progeny plant selected by the method of claim 9 wherein the plant has at least one allele of a marker locus wherein the marker locus is located within a chromosomal interval comprising and flanked by PZE-103082924 and PZE-103101055. 